Preparation of the enzyme beta-glucanase by fermentation of fungi

ABSTRACT

β-glucanase is produced under submerged, aerobic and thermophilic conditions, by cultivating the microorganism Rhizomucor pusillus (Lindt) Schipper, preferably the strain CBS 551.82. Fermentation is carried out using a fed batch method, where a carbon source such as barley meal is first introduced, and when this is consumed, a sugar compound, preferably lactose, is added, whereupon the fermentation continues until the carbon source is completely or partially consumed.

This invention concerns a procedure for the production of β-glucanasethrough the cultivation of a microorganism in a nutrient medium underoptimal conditions.

We refer to Norwegian patent application No. 82 4321 concerning the useof microorganisms of the species Rhizomucor pusillus (Lindt) Schipper asbeing suitable for the above process, as well as an isolated andregistered strain of the stated microorganism CBS 551.82 (Centraalbureauvoor Schimmelcultures, Nederland) being particularly suitable for theproduction of the enzyme β-glucanase.

The purpose of this invention is to optimize fermentation such thatmaximum enzyme yield within the shortest possible time is achieved, yetunder the same conditions as otherwise stated in Norwegian patentapplication No. 82 4321, that is, to conduct fermentation underthermophilic conditions and wherein the microorganism is thermostableunder the said conditions. In addition to the significance that themicroorganism (Rhizomucor pusillus (Lindt) Schipper CBS 551.87) has fora positive result, it should also be mentioned that the composition ofthe nutrient medium, the air delivery rate and apparatus design are allimportant factors for the achievement of optimal conditions forfermentation.

Vegetable meal and starchy substances, as well as sugars, are commonlyused as carbon sources for fermentation purposes. However, not allcarbon sources have proven to be suitable for this purpose. The cause ofthis has not been definitely ascertained, but different factors appearto have an influence on the transformation of sugar into energy. If thesource of carbon is to be used as an energy source, the followingfactors at least seem to be of significance: the solubility of thesource of carbon and its ability to be taken up through the system ofcell wall and membrane.

It now appears, astonishingly enough, that if we apply a two-stagefermentation with lactose as the source of carbon in a fermentationprocess using the previously mentioned microorganisms, then the enzymeyield increases several times when all conditions are met.

Under batch conditions, the fermentation exhibits the traditional growthpattern with a lag phase, exponential growth and a stationary phasewhere enzyme production is greatest in the stationary phase. It isimportant that the fermentation is carried out under aerobic conditions.This places great demands on the stirring machinery and on the stirringspeed. If the stirring is too vigorous, then the fungal hyphae(microorganisms) are disintegrated, and if it is too weak, oxygen is notdistributed sufficiently in the fermentor. Both factors reduce enzymeformation. Furthermore, too vigorous stirring combined with a low oxygencontent promotes a threadlike structure in the fungal hyphae, whereas amore pellet-like structure is desired.

For a better understanding of the invention we refer to Examples 1-8.

EXAMPLE 1

(a) 300 ml of an inoculum of Rhizomucor pusillus (Lindt) Schipper CBS551.82 was mixed in a 14 l fermentor with 10 l of a salt medium of thefollowing composition:

KH₂ PO₄ : 2.00 g/l

MgSO₄ . 7 H₂ O: 1.75 g/l

CaCl₂ : 0.20 g/l

Trace mineral solution: 2.5 ml/l

Water: 1.0 l

The trace mineral solution has the following composition:

CuSo₄ . 5 H₂ O: 1.0 g/l

FeSO₄ . 7 H₂ O: 15.0 g/l

ZnSO₄ . 7 H₂ O: 6.2 g/l

MnSO₄ . H₂ O: 1.5 g/l

Conc. H₂ SO₄ : 1.5 ml/l

The fermentation was carried out with 40 g/l barley meal as the carbonsource and 13.4 g/l NH₄ Cl as the nitrogen source. Berol 374 from BerolKemi AB, Sweden, was used as an anti-foaming agent. The stirring ratewas 600 rpm, the temperature 40° C. and pH 4.5. The fermentor's airdelivery was set to 3 vvm, that is, 30 l air/min. (1 vvm is 1 1 air per1 nutrient medium per min.). Fermentation time was 45 hours. (b) Aninoculum of the above mentioned 10 l nutrient medium was mixed in a 300l fermentor with 200 l of the salt medium described above.

Fermentation was carried out according to the batch method, with amixture of 40 g/l barley meal and 13.4 g/l NH₄ Cl at pH 4.5 and atemperature of 40° C., and a stirring rate of 410 rpm. The fermentor'sair delivery rate was 0.2 vvm, or 40 l air/min. The enzyme activity was2.5 EU/ml after 79 hours and 2.7 EU/ml after 92 hours according toDygert's method (Analytical Biochemistry, vol. 13 (1965) pp. 367-374).

EXAMPLE 2

An inoculum from a 14 l fermentor holding the conditions described inExample 1 was put into a 300 l fermentor in a quantity of 9 l andcultivated using the batch method with 20, 30, 40 and 50 g/l barley mealand fermented as in Example 1b.

As the following table shows, enzyme activity changed with changingbarley meal concentrations:

20 g barley meal/l: 2.00 IRV/ml

30 g barley meal/l: 6.50 IRV/ml

40 g barley meal/l: 7.50 IRV/ml

50 g barley meal/l: 1.50 IRV/ml

IRV is the "Increase in Reciprocal Viscosity". This can be compraed withenzyme activity (EU/ml) as measured by Dygert's method using thefollowing formula:

    IRV/ml=EU/ml×2.5

Otherwise we refer to J. Inst. Brewing, Vol. 85 (1979), pp. 92-94, wherethe capillary-viscosimetric method, giving the result in terms of IRVunits, is discussed.

EXAMPLE 3

An inoculum from a 14 l fermentor holding the cultivating conditionsdescribed in Example 1a, was put into a 300 l fermentor in a quantity of9 l and cultivated according to the fed batch method, using 15 g/lbarley meal in the beginning stage and NH₄ Cl in a concentration of 1.38g/l. When the barley meal was consumed, glucose was added at a feed rateof 1.5 g/min. with a total C-source consumption of 40 g/l. The pH wasadjusted to 4.5 using an equivalent mixture of NH₃ and NH₄ Cl, and thefermentation was carried out under constant temperature conditions at40° C. The stirring rate was 410 rpm and the fermentor's air deliveryrate was 40 l/min. Berol 374 was used as a foaming inhibitor. Fermentingtime was 110 h. Enzyme activity according to the capillary-viscosimetricmethod was 11.1 IRV/ml.

EXAMPLE 4

An inoculum from a 14 l fermentor holding the conditions described inExample 1b was put into a 300 l fermentor in a quantity of 9 l andcultivated according to the fed batch method, as described in Example 3,with the exception that 25 g lactose/l was used instead of glucose.Fermenting time: 95 hours. Enzyme activity according to thecapillary-viscosimetric method was 30.4 IRV/ml.

EXAMPLE 5

The procedure was the same as in Example 4, except that the lactosequantity was increased to 35 g lactose/l. Fermenting time: 95 hours.Enzyme activity according to the capillary-viscosimetric method was 38.0IRV/ml.

EXAMPLE 6

The procedure was the same as in Example 4, except that sucrose was usedas the carbon source. Enzyme activity was measured as 2.6 IRV/ml.

EXAMPLE 7

Lactose only was used as a carbon source for the fermentation. Thefermentation was otherwise carried out as described in Example 1. Enzymeactivity was measured as 8.2 IRV/ml.

EXAMPLE 8

The procecure was the same as in Example 4, but raffinose was used asthe carbon source. Enzyme activity was measured as 6.7 IRV/ml.

In the examples that have been discussed, both a simple "batch" methodand a "fed batch" method, as described in Examples 1,2 and 3, 4, 5, 6, 7and 8 respectively, have been used. The batch method usually yields afungal hyphae with a threadlike structure, which among other thingsimpedes filtering. The fed batch method, however, produces the fungalhyphae in fine-grained, homogeneous pellets, which is preferable forreasons concerning process technology.

The cause of this favourable operational result seems to rest with thefact that in the fed batch method, the fungal hyphae is limited by thecarbohydrate supply rather than by the oxygen supply, as with the simplebatch method.

Otherwise we refer to FIG. 1 which shows the increase in enzyme activityin relation to fermenting time.

Curves D, E and F show the increase in enzyme activity when fermentationis carried out as in Examples 1, 3 and 4.

We claim:
 1. A process for producing β-glucanase under submerged,aerobic and thermophilic conditions, which comprises cultivating in anutrient medium a fungal strain of Rhizomucor pusillus (Lindt) Schipper,wherein an inoculum of said strain is introduced into a fermentorcontaining a nutrient medium and fermentation occurs according to a fedbatch method, where a starchy vegetable meal is added first as a sourceof carbon, and when said starchy vegetable meal is completely orpartially consumed, a sugar compound selected from the group consistingof lactose, glucose, sucrose and raffinose is then introduced as asource of carbon, whereupon the fermentation continues until the carbonsource is completely or partially consumed.
 2. The process according toclaim 1, wherein the fungal strain is Rhizomucor pusillus (Lindt)Schipper CBS 551.82.
 3. The process according to claim 1, wherein thestarchy vegetable meal is barley meal.
 4. The process according to claim1, wherein the sugar compound is lactose.